Suppression of specific classes of soybean seed protein genes

ABSTRACT

This invention concerns the construction of transgenic soybean lines wherein the expression of genes encoding seed storage proteins are modulated to effect a change in seed storage protein profile of transgenic plants. Modification of the seed storage protein profile can result in the production of novel soy protein products with unique and valuable functional characteristics.

This is a continuation of PCT/US97/09743, filed Jun. 10, 1997 and whichclaims priority to U.S. Provisional Application No. 60/019,940, filedJun. 14, 1996.

FIELD OF THE INVENTION

This invention concerns the construction of transgenic soybean lineswherein the expression of genes encoding seed storage proteins ismodified to effect a change in seed storage protein profile oftransgenic plants. Such modified transgenic soybean lines are used forthe production of novel soy protein products with unique and valuablefunctional characteristics.

BACKGROUND OF THE INVENTION

Soybean seeds contain from 35% to 55% protein on a dry weight basis. Themajority of this protein is storage protein, which is hydrolyzed duringgermination to provide energy and metabolic intermediates needed by thedeveloping seedling. The soybean seed's storage protein is an importantnutritional source when harvested and utilized as a livestock feed. Inaddition, it is now generally recognized that soybeans are the mosteconomical source of protein for human consumption. Soy protein orprotein isolates are already used extensively for food products indifferent parts of the world. Much effort has been devoted to improvingthe quantity and quality of the storage protein in soybean seeds.

The seeds of most plant species contain what are known in the art asseed storage proteins. These have been classified on the basis of theirsize and solubility (Higgins, T. J. (1984)Ann. Rev. Plant Physiol.35:191-221). While not every class is found in every species, the seedsof most plant species contain proteins from more than one class.Proteins within a particular solubility or size class are generally morestructurally related to members of the same class in other species thanto members of a different class within the same species. In manyspecies, the seed proteins of a given class are often encoded bymultigene families, sometimes of such complexity that the families canbe divided into subclasses based on sequence homology.

There are two major soybean seed storage proteins:glycinin (also knownas the 11S globulins) and β-conglycinin (also known as the 7Sglobulins). Together, they comprise 70 to 80% of the seed's totalprotein, or 25 to 35% of the seed's dry weight. Glycinin is a largeprotein with a molecular weight of about 360 kDa. It is a hexamercomposed of the various combinations of five major isoforms (commonlycalled subunits) identified as G1, G2, G3, G4 and G5. Each subunit is inturn composed of one acidic and one basic polypeptide held together by adisulfide bond. Both the acidic and basic polypeptides of a singlesubunit are coded for by a single gene. Hence, there are fivenon-allelic genes that code for the five glycinin subunits. These genesare designated Gy1, Gy2, Gy3, Gy4 and Gy5, corresponding to subunits G1,G2, G3, G4 and G5, respectively (Nielsen, N. C. et al. (1989) Plant Cell1:313-328).

Genomic clones and cDNA's for glycinin subunit genes have been sequencedand fall into two groups based on nucleotide and amino acid sequencesimilarity. Group I consists of Gy1, Gy2, and Gy3, whereas Group IIconsists of Gy4 and Gy5. There is greater than 85% similarity betweengenes within a group (i.e., at least 85% of the nucleotides of Gy1, Gy2and Gy3 are identical, and at least 85% of the nucleotides of Gy4 andGy5 are identical), but only 42% to 46% similarity between the genes ofGroup I and Group II.

β-Conglycinin (a 7S globulin) is a heterogeneous glycoprotein with amolecular weight ranging from 150 and 240 kDa. It is composed of varyingcombinations of three highly negatively charged subunits identified asα, α′ and β. cDNA clones representing the coding regions of the genesencoding the the α and α′ subunits have been sequenced and are ofsimilar size; sequence identity is limited to 85%. The sequence of thecDNA representing the coding region of the β subunit, however, is nearly0.5 kb smaller than the α and α′ cDNAs. Excluding this deletion,sequence identity to the α and α′ subunits is 75-80%. The three classesof β-conglycinin subunits are encoded by a total of 15 subunit genesclustered in several regions within the genome soybean (Harada, J. J. etal. (1989) Plant Cell 1:415-425).

New soy based products such as protein concentrates, isolates, andtextured protein products are increasingly utilized in countries that donot necessarily accept traditional oriental soy based foods. Use ofthese new products in food applications, however, depends on localtastes and functional characteristic of the protein products relative torecipe requirements. Over the past 10 years, significant effort has beenaimed at understanding the functional characteristics of soybeanproteins. Examples of functional characteristics include water sorptionparameters, wettability, swelling, water holding, solubility,thickening, viscosity, coagulation, gelation characteristics andemulsification properties. A large portion of this body of research hasfocused on study of the β-conglycinin and glycinin proteinsindividually, as well as how each of these proteins influences the soyprotein system as a whole (Kinsella, J. E. et al. (1985) New ProteinFoods 5:107-179; Morr, C. V. (1987) JAOCS 67:265-271; Peng, L. C. et al.(1984) Cereal Chem 61:480-489). Because functional properties aredirectly related to physiochemical properties of proteins, thestructural differences of β-conglycinin and glycinin result in these twoproteins having significantly different functional characteristics.Differences in thermal aggregation, emulsifying properties, and waterholding capacity have been reported. In addition, gelling propertiesvary as well, with glycinin forming gels that have greater tensilestrain, stress, and shear strength, better solvent holding capacity, andlower turbidity. However, soy protein products produced today are ablend of both glycinin and β-conglycinin and therefore have functionalcharacteristics dependent on the blend of glycinin's and β-conglycinin'sindividual characteristics. For example, when glycinin is heated to 100°C., about 50% of the protein is rapidly converted into solubleaggregates. Further heating results in the enlargement of the aggregatesand in their precipitation. The precipitate consists of the glycinin'sbasic polypeptides; the acidic polypeptides remain soluble. The presenceof β-conglycinin inhibits the precipitation of the basic polypeptides byforming soluble complexes with them. Whether heat denaturation isdesireable or not depends on the intended use. If one could produce soyprotein products containing just one or the other storage protein,products requiring specific physical characteristics derived fromparticular soy proteins would become available or would be moreeconomical to produce.

Over the past 20 years, soybean lines lacking one or more of the variousstorage protein subunits (null mutations) have been identified in thesoybean germplasm or produced using mutational breeding techniques.Breeding efforts to combine mutational events have resulted in soybeanlines whose seeds contain about half the normal amount of β-conglycinin(Takashashi, K. et al. (1994) Breeding Science 44:65-66; Kitamura, J.(1995) JARQ 29:1-8). The reduction of β-conglycinin is controlled bythree independent recessive mutations. Recombining glycinin subunit nullmutations have resulted in lines whose seeds have significantly reducedamounts of glycinin (Kitamura, J. (1995) JARQ 29:1-8). Again, reductionis controlled by three independent recessive mutations. Developingagronomically viable soybean varieties from the above lines, in whichthe seed contains only glycinin or β-conglycinin, will be time consumingand costly. Each cross will result in the independent segregation of thethree mutational events. In addition, each mutational event will need tobe in the homozygous state. Development of high yielding agronomicallysuperior soybean lines will require the screening and analysis of alarge number of progeny over numerous generations.

Antisense technology has been used to reduce specific storage proteinsin seeds. In Brassica napus, napin (a 2S albumin) and cruciferin (an 11Sglobulin) are the two major storage proteins, comprising about 25% and60% of the total seeds protein, respectively. Napin proteins are codedfor by a large multi-gene family of up to 16 genes; several cDNA andgenomic clones have been sequenced (Josefsson, L.-G. et al. (1987) J.Biol Chem 262:12196-12201; Schofield, S. and Crouch, M. L. (1987) J.Biol. Chem. 262:12202-12208). The genes exhibit greater than 90%sequence identity in both their coding and flanking regions. Thecruciferin gene family is equally complex, comprising 3 subfamilies witha total of 8 genes (Rodin, J. et al. (1992) Plant Mol. Biol.20:559-563). Kohno-Murase et al. ((1994) Plant Mol. Biol. 26:1115-1124)demonstrated that a napin antisense gene using the napA gene driven bythe napA promoter could be used to construct transgenic plants whoseseeds contained little or no napin.

The same group (Kohno-Murase et al. (1995) Theoret. Applied Genetics91:627-631) attempted to reduce cruciferin (11S globulin) expression inBrassica napus by expressing an antisense form of a cruciferin gene(cruA, encoding an alpha 2/3 isoform) under the control of the napApromoter. In this case the results were more complex. The cruciferinsare divided into three subclasses based on sequence identity (alpha 1,2/3, and 4); the classes each have from 60-75% sequence identity witheach other (Rodin, J. et al. (1992) Plant Mol Biol. 20:559-563).Expression of the antisense gene encoding the alpha 2/3 isoform resultedin lower levels of the alpha 1 and 2/3 forms. However, there was noreduction in the expression of the alpha 4 class.

Antisense technology was used to reduce the level of the seed storageprotein, glutelin, in rice. Expression of the seed specific glutelinpromoter operably linked to the full length antisense glutelin codingregion resulted in about a 25% reduction in glutelin protein levels(U.S. Pat. No. 5,516,668).

SUMMARY OF THE INVENTION

The instant invention provides a method for reducing the quantityglycinin or β-conglycinin (11S or 7S globulins, respectively) seedstorage proteins in soybeans. In one embodiment, cosuppressiontechnology was used to suppress the expression of genes encoding the7S-globulin class of seed protein genes. Genes encoding either two (αand α′) or all three subclasses (α, α′ and β) of 7S globulins weresuppressed by expression of the gene encoding a single subclass (α) ofβ-conglycinin, resulting in soybean lines with altered seed storageprofiles. In another embodiment, a method for suppressing two completelydifferent genes, only one of which is a seed protein gene, is presented,allowing for multiple changes in seed composition. Surprisingly,expression of a chimeric gene comprising the promoter region of asoybean seed storage protein operably linked to the coding region of asoybean gene whose expression alters the fatty acid profile oftransgenic soybean seeds resulted in simultaneous alteration of twodistinct phenotypic traits: seed storage protein profile and seed oilprofile.

The method for reducing the quantity of soybean seed storage proteintaught herein comprises the following steps:

(a) constructing a chimeric gene comprising (i) a nucleic acid fragmentencoding a promoter that is functional in the cells of soybean seeds,(ii) a nucleic acid fragment encoding all or a portion of a soybean seedstorage protein placed in sense or antisense orientation relative to thepromoter of (i), and (iii) a transcriptional termination region;

(b) creating a transgenic soybean cell by introducing into a soybeancell the chimeric gene of (a); and

(c) growing the transgenic soybean cells of step (b) under conditionsthat result in expression of the chimeric gene of step (a) wherein thequantity of one or more members of a class of soybean seed storageprotein subunits is reduced when compared to soybeans not containing thechimeric gene of step (a).

DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF THE SEQUENCEDESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the Sequence Descriptions which form apart of thisapplication. The Sequence Descriptions contain the three letter codesfor amino acids as defined in 37 C.F.R. 1.822 which are incorporatedherein by reference.

SEQ ID NO:1 shows the 5′ to 3′ nucleotide sequence encoding the αsubunit of the β-conglycinin soybean seed storage protein.

SEQ ID NO:2 shows the 5′ to 3′ nucleotide sequence encoding the α′subunit of the β-conglycinin soybean seed storage protein.

SEQ ID NO:3 shows the 5′ to 3′ nucleotide sequence encoding the βsubunit of the β-conglycinin soybean seed storage protein.

SEQ ID NOS:4 and 5 show the nucleotide sequences of the PCR primers ConSand Con1.4a (respectively) used to isolate nucleic acid fragmentsencoding the α and α′ subunits of the β-conglycinin soybean seed storageprotein.

SEQ ID NOS:6 and 7 show nucleotide sequences of the PCR primers Con.09and Con.8 (respectively) used to distinguish nucleic acid fragmentsencoding the α and α′ subunits of the β-conglycinin soybean seed storageprotein.

SEQ ID NOS:8 and 9 show the nucleotide sequences of the PCR primersConSa and Con1.9a (respectively) used to isolate full length cDNAsencoding the α and α′ subunits of the β-conglycinin soybean seed storageprotein.

SEQ ID NO:10 shows the nucleotide sequence of the PCR primer Con.1.0used to confirm the full length cDNA encoding the α and α′ subunits ofthe β-conglycinin soybean seed storage protein.

SEQ ID NOS:11, 12 and 13 show the 5′ to 3′ nucleotide sequences encodingthe Gy1, Gy2 and Gy3 subunits (respectively) of the group I glycininsoybean seed storage protein.

SEQ ID NOS:14 and 15 show the 5′ to 3′ nucleotide sequences encoding theGy4 and Gy5 subunits (respectively) of the group II glycinin soybeanseed storage protein.

SEQ ID NOS:16, 17 and 18 show the nucleotide sequences of the PCRprimers G1-1, G1-1039 and G1-1475 (respectively) used to isolate thecDNAs encoding the subunits of the group I glycinin soybean seed storageprotein.

SEQ ID NOS:19, 20 and 21 show the nucleotide sequences of the PCRprimers G4-7, G4-1251, and G4-1670 (respectively) used to isolate thecDNA encoding the subunits of the group II glycinin soybean seed storageprotein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a restriction map of plasmid pML70, used as an intermediatecloning vehicle in construction of chimeric genes of the instantinvention.

FIG. 2 is a restriction map of plasmid pCW109, used as an intermediatecloning vehicle in construction of chimeric genes of the instantinvention.

FIG. 3 is a restriction map of plasmid pKS18HH, used as an intermediatecloning vehicle in construction of chimeric genes of the instantinvention.

FIG. 4 is a restriction map of plasmid pJo1. This plasmid was derived bycloning the plant transcriptional unit KTi promoter/truncated α subunitof β-conglycinin/KTi 3′ end into the BamH I site of pKS18HH.

FIG. 5 is an SDS-PAGE gel of extracted protein from somatic embryostransformed with pJo1.

FIG. 6 is a restriction map of plasmid pBS43. This plasmid comprises anucleic acid sequence encoding the Glycine max microsomal delta-12desaturase under the transcriptional control of the soybeanβ-conglycinin promoter.

FIG. 7 is an SDS-PAGE gel of extracted protein from soybean seedsobtained from plants transformed with pBS43.

FIG. 8 is a restriction map of plasmid pJo3. This plasmid was derived bycloning the plant transcriptional unit KTi promoter/full length cDNA ofthe α subunit of β-conglycinin/KTi 3′ end into the HindIII site ofpKS18HH.

FIG. 9 is a restriction map of plasmid pRB20. This plasmid was derivedby cloning the transcriptional unit β-conglycinin promoter/Phaseolin 3′end into the HindIII site of pKS18HH. It is used as an intermediatecloning vehicle in construction of chimeric genes of the instantinvention.

BIOLOGICAL DEPOSITS

The following plasmids have been deposited under the terms of theBudapest Treaty at American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, and bear the followingaccession numbers:

Plasmid Accession Number Date of Deposit pJol ATCC 97614 June 15, 1996pBS43 ATCC 97619 June 19, 1996 pJo3 ATCC 97615 June 15, 1996

DEFINITIONS

In the context of this disclosure, a number of terms shall be used. Theterm “nucleic acid” refers to a large molecule which can besingle-stranded or double-stranded, composed of monomers (nucleotides)containing a sugar, a phosphate and either a purine or pyrimidine. A“nucleic acid fragment” is a fraction of a given nucleic acid molecule.In higher plants, deoxyribonucleic acid (DNA) is the genetic materialwhile ribonucleic acid (RNA) is involved in the transfer of theinformation in DNA into proteins. A “genome” is the entire body ofgenetic material contained in each cell of an organism. The term“nucleotide sequence” refers to the sequence of DNA or RNA polymers,which can be single-or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases capable of incorporation intoDNA or RNA polymers.

As used herein, the term “homologous to” refers to the relatednessbetween the nucleotide sequence of two nucleic acid molecules or betweenthe amino acid sequences of two protein molecules. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.); or by the comparison of sequence similaritybetween two nucleic acids or proteins, such as by the method ofNeedleman et al. ((1970) J. Mol. Biol. 48:443-453).

As used herein, “essentially similar” refers to DNA sequences that mayinvolve base changes that do not cause a change in the encoded aminoacid, or which involve base changes which may alter one or more aminoacids, but do not affect the functional properties of the proteinencoded by the DNA sequence. It is therefore understood that theinvention encompasses more than the specific exemplary sequences.Modifications to the sequence, such as deletions, insertions, orsubstitutions in the sequence which produce silent changes that do notsubstantially affect the functional properties of the resulting proteinmolecule are also contemplated. For example, alteration in the genesequence which reflect the degeneracy of the genetic code, or whichresults in the production of a chemically equivalent amino acid at agiven site, are contemplated; thus, a codon for the amino acid alanine,a hydrophobic amino acid, may be substituted by a codon encoding anotherhydrophobic amino acid residue such as glycine, valine, leucine, orisoleucine. Similarly, changes which result in substitution of onenegatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a biologicallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the protein molecule would alsonot be expected to alter the activity of the protein. In some cases, itmay in fact be desirable to make mutants of the sequence in order tostudy the effect of alteration on the biological activity of theprotein. Each of the proposed modifications is well within the routineskill in the art, as is determination of retention of biologicalactivity of the encoded products. Moreover, the skilled artisanrecognizes that “essentially similar” sequences encompassed by thisinvention can also defined by their ability to hybridize, understringent conditions (0.1×SSC, 0.1% SDS, 65° C.), with the sequencesexemplified herein.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-coding) andfollowing (3′ non-coding) the coding region. “Native” gene refers to anisolated gene with its own regulatory sequences as found in nature.“Chimeric gene” refers to a gene that comprises heterogeneous regulatoryand coding sequences not found in nature. “Endogenous” gene refers tothe native gene normally found in its natural location in the genome andis not isolated. A “foreign” gene refers to a gene not normally found inthe host organism but that is introduced by gene transfer. “Codingsequence” or “coding region” refers to a DNA sequence that codes for aspecific protein and excludes the non-coding sequences. It mayconstitute an “uninterrupted coding sequence”, i.e., lacking an intronor it may include one or more introns bounded by appropriate splicejunctions. An “intron” is a nucleotide sequence that is transcribed inthe primary transcript but that is removed through cleavage andre-ligation of the RNA within the cell to create the mature mRNA thatcan be translated into a protein. “Initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides in a codingsequence that specifies initiation and chain termination, respectively,of protein synthesis (niRNA translation). “Open reading frame” refers tothe coding sequence uninterrupted by introns between initiation andtermination codons that encodes an amino acid sequence. “RNA transcript”refers to the product resulting from RNA polymerase-catalyzedtranscription of a DNA sequence. When the RNA transcript is a perfectcomplementary copy of the DNA sequence, it is referred to as the primarytranscript or it may be a RNA sequence derived from posttranscriptionalprocessing of the primary transcript and is referred to as the matureRNA. “Messenger RNA (mRNA)” refers to the RNA that is without intronsand that can be translated into protein by the cell. “cDNA” refers to adouble-stranded DNA that is complementary to and derived from mRNA.“Sense” RNA refers to RNA transcript that includes the mRNA. “AntisenseRNA” refers to a RNA transcript that is complementary to all or part ofa target primary transcript or mRNA and that blocks the expression of atarget gene. The complementarity of an antisense RNA may be with anypart of the specific gene transcript, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence.

As used herein, “suitable regulatory sequences” refer to nucleotidesequences in native or chimeric genes that are located upstream (5′),within, or downstream (3′) to the nucleic acid fragments of theinvention, which control the expression of the nucleic acid fragments ofthe invention. The term “expression”, as used herein, refers to thetranscription and stable accumulation of the sense (mRNA) or theantisense RNA derived from the nucleic acid fragment(s) of the inventionthat, in conjunction with the protein apparatus of the cell, results inaltered phenotypic traits. Expression of the gene involves transcriptionof the gene and translation of the mRNA into precursor or matureproteins. “Antisense inhibition” refers to the production of antisenseRNA transcripts capable of preventing the expression of the targetprotein. “Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Cosuppression” refers to the expression of aforeign gene which has substantial homology to an endogenous generesulting in the suppression of expression of both the foreign and theendogenous gene. “Altered levels” refers to the production of geneproduct(s) in transgenic organisms in amounts or proportions that differfrom that of normal or non-transformed organisms. The skilled artisanwill recognize that the phenotypic effects contemplated by thisinvention can be achieved by alteration of the level of gene product(s)produced in transgenic organisms relative to normal or non-transformedorganisms, namely a reduction in gene expression mediated by antisensesuppression or cosuppression.

“Promoter” refers to a DNA sequence in a gene, usually upstream (5′) toits coding sequence, which controls the expression of the codingsequence by providing the recognition for RNA polymerase and otherfactors required for proper transcription. In artificial DNA constructs,promoters can also be used to transcribe antisense RNA. Promoters mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions. It may alsocontain enhancer elements. An “enhancer” is a DNA sequence which canstimulate promoter activity. It may be an innate element of the promoteror a heterologous element inserted to enhance the level ortissue-specificity of a promoter. “Constitutive promoters” refers tothose that direct gene expression in all tissues and at all times.“Tissue-specific” or “development-specific” promoters as referred toherein are those that direct gene expression almost exclusively inspecific tissues, such as leaves or seeds, or at specific developmentstages in a tissue, such as in early or late embryogenesis,respectively.

The “3′ non-coding sequences” refers to the DNA sequence portion of agene that contains a polyadenylation signal and any other regulatorysignal capable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor.

The term “operably linked” refers to nucleic acid sequences on a singlenucleic acid molecule which are associated so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a structural gene when it is capable of affecting the expression ofthat structural gene (i.e., that the structural gene is under thetranscriptional control of the promoter).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritence. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

This invention concerns the construction of transgenic soybean lineswherein the expression of genes encoding seed storage proteins aremodulated to effect a change in seed storage protein profile oftransgenic plants. Modification of the seed storage protein profile canresult in production of novel soy protein products with unique andvaluable functional characteristics.

Gene expression in plants uses regulatory sequences that are functionalin such plants. The expression of foreign genes in plants iswell-established (De Blaere et al. (1987) Meth. Enzymol. 153:277-291).The source of the promoter chosen to drive the expression of thefragments of the invention is not critical provided it has sufficienttranscriptional activity to accomplish the invention by decreasing theexpression of the target seed storage protein genes.

Preferred promoters include strong constitutive plant promoters, such asthose directing the 19S and 35S transcripts in cauliflower mosaic virus(Odell, J. T. et al. (1985) Nature 313:810-812; Hull et al. (1987)Virology 86:482-493).

Particularly preferred promoters are those that allow seed-specificexpression. Examples of seed-specific promoters include, but are notlimited to, the promoters of seed storage proteins, which can representup to 90% of total seed protein in many plants. The seed storageproteins are strictly regulated, being expressed almost exclusively inseeds in a highly tissue-specific and stage-specific manner (Higgins etal. (1984) Ann. Rev. Plant Physio. 35:191-221; Goldberg et al. (1989)Cell 56:149-160). Moreover, different seed storage proteins may beexpressed at different stages of seed development.

Expression of seed-specific genes has been studied in great detail (Seereviews by Goldberg et al. (1989) Cell 56:149-160 and Higgins et al.(1984) Ann. Rev. Plant Physiol. 35:191-221). There are currentlynumerous examples of seed-specific expression of seed storage proteingenes (natural or chimeric) in transgenic dicotyledonous plants; ingeneral, temporal and spatial expression patterns are maintained. Thepromoters used in such examples could potentially be used to affect thepresent invention. These include genes from dicotyledonous plants forbean β-phaseolin (Sengupta-Gopalan et al.(1985) Proc. Natl. Acad. Sci.USA 82:3320-3324; Hoffman et al. (1988) Plant Mol. Biol. 11:717-729),bean lectin (Voelker et al. (1987) EMBO J. 6:3571-3577), soybean lectin(Okamuro et al. (1986) Proc. Natl. Acad. Sci. USA 83:8240-8244), soybeanKunitz trypsin inhibitor (Perez-Grau et al. (1989) Plant Cell1:095-1109), soybean β-conglycinin (Beachy et al. (1985) EMBO J.4:3047-3053; pea vicilin (Higgins et al. (1988) Plant Mol. Biol.11:683-695), pea convicilin (Newbigin et al. (1990) Planta 180:461-470),pea legumin (Shirsat et al. (1989) Mol. Gen. Genetics 215:326-331),rapeseed napin (Radke et al. (1988) Theor. Appl. Genet. 75:685-694) andArabidopsis thaliana 2S albumin (Vandekerckhove et al. (1989)Bio/Technology 7:929-932).

Of particular use in the expression of the nucleic acid fragment of theinvention will be the heterologous promoters from several soybean seedstorage protein genes such as those for the Kunitz trypsin inhibitor(KTi; Jofuku et al. (1989) Plant Cell 1:1079-1093; glycinin (Nielson etal. (1989) Plant Cell 1:313-328), and β-conglycinin (Harada et al.(1989) Plant Cell 1:415-425). The skilled artisan will recognize thatattention must be paid to differences in temporal regulation endowed bydifferent seed promoters. For example, the promoter for the α-subunitgene is expressed a few days before that for the β-subunit gene (Beachyet al. (1985) EMBO J. 4:3047-3053), so that the use of the β-subunitgene is likely to be less useful for suppressing α-subunit expression.

Also of potential use, but less preferred, will be the promoters ofgenes involved in other aspects of seed metabolism, such as lipid orcarbohydrate biosynthesis. In summary, the skilled artisan will have nodifficulty in recognizing that any promoter of sufficient strength andappropriate temporal expression pattern can potentially be used toimplement the present invention. Similarly, the introduction ofenhancers or enhancer-like elements into the promoter regions of eitherthe native or chimeric nucleic acid fragments of the invention wouldresult in increased expression to accomplish the invention. This wouldinclude viral enhancers such as that found in the 35S promoter (Odell etal. 1988) Plant Mol. Biol. 10:263-272), enhancers from the opine genes(Fromm et l. (1989) Plant Cell 1:977-984), or enhancers from any othersource that result in increased transcription when placed into apromoter operably linked to the nucleic acid fragment of the invention.

Of particular importance is the DNA sequence element isolated from thegene encoding the α-subunit of β-conglycinin that can confer a 40-fold,seed-specific enhancement to a constitutive promoter (Chen et al. (1989)Dev. Genet. 10:112-122). One skilled in the art can readily isolate thiselement and insert it within the promoter region of any gene in order toobtain seed-specific enhanced expression with the promoter in transgenicplants. Insertion of such an element in any seed-specific gene that isnormally expressed at times different than the β-conglycinin gene willresult in expression of that gene in transgenic plants for a longerperiod during seed development.

Any 3′ non-coding region capable of providing a polyadenylation signaland other regulatory sequences that may be required for the properexpression of the nucleic acid fragments of the invention can be used toaccomplish the invention. This would include 3′ ends of the native fattyacid desaturase(s), viral genes such as from the 35S or the 19Scauliflower mosaic virus transcripts, from the opine synthesis genes,ribulose 1,5-bisphosphate carboxylase, or chlorophyll a/b bindingprotein. There are numerous examples in the art that teach theusefulness of different 3′ non-coding regions.

Various methods of transforming cells of higher plants according to thepresent invention are available to those skilled in the art (seeEuropean Patent Publications EP-A-295,959 and EP-A-318,341). Suchmethods include those based on transformation vectors utilizing the Tiand Ri plasmids of Agrobacterium spp. It is particularly preferred touse the binary type of these vectors. Ti-derived vectors transform awide variety of higher plants, including monocotyledonous anddicotyledonous plants (Sukhapinda et al. (1987) Plant Mol Biol.8:209-216; Potrykus, (1985) Mol. Gen. Genet. 199:183). Othertransformation methods are available to those skilled in the art, suchas direct uptake of foreign DNA constructs (see European PatentPublication EP-A-295,959), techniques of electroporation (Fromm et al.(1986) Nature (London) 319:791) or high-velocity ballistic bombardmentwith metal particles coated with the nucleic acid constructs (Klein etal. (1987) Nature (London) 327:70). Once transformed, the cells can beregenerated by those skilled in the art. Of particular relevance are therecently described methods to transform soybean, including McCabe et al.((1988) Bio/Technology 6:923-926), Finer et al. ((1991) In Vitro Cell.Dev. Biol. 27:175-182) and Hinchee, M. A. W. ((1988) Bio/Technology6:915-922).

Once transgenic plants are obtained by one of the methods describedabove, it is necessary to screen individual transgenics for those thatmost effectively display the desired phenotype. It is well known tothose skilled in the art that individual transgenic plants carrying thesame construct may differ in expression levels; this phenomenon iscommonly referred to as “position effect”. Thus, in the presentinvention different individual transformants may vary in theeffectiveness of suppression of the target seed protein. The personskilled in the art will know that special considerations are associatedwith the use of antisense or cosuppression technologies in order toreduce expression of particular genes. U.S. Pat. Nos. 5,190,931,5,107,065 and 5,283,323 have taught the feasibility of these techniques,but it is well known that their efficiency is unpredictable.Accordingly, the person skilled in the art will make multiple geneticconstructs containing one or more different parts of the gene to besuppressed, since the art does not teach a method to predict which willbe most effective for a particular gene. Furthermore, even the mosteffective constructs will give an effective suppression phenotype onlyin a fraction of the individual transgenic lines isolated. For example,World Patent Publications WO93/11245 and WO94/11516 teach that whenattempting to suppress the expression of fatty acid desaturase genes incanola, actual suppression was obtained in less than 1% of the linestested. In other species the percentage is somewhat higher, but in nocase does the percentage reach 100. This should not be seen as alimitation on the present invention, but instead as practical matterthat is appreciated and anticipated by the person skilled in this art.Accordingly, the skilled artisan will develop methods for screeninglarge numbers of transformants. The nature of these screens willgenerally be chosen on practical grounds, and is not an inherent part ofthe invention. A preferred method will be one which allows large numbersof samples to be processed rapidly, since it will be expected that themajority of samples will be negative.

The mechanism of cosuppression remains unclear (for one review andspeculation, see Flavell, R. (1994) Proc. Natl. Acad Sci. USA91:3490-3496), and therefore the exact requirements to induce it whendesired are also unclear. Most examples found in the literature involvethe use of all or a large part of the transcribed region of the gene tobe cosuppressed to elicit the desired response. However, in at least onecase (Brusslan et al. (1993) Plant Cell 5:667-677; Brusslan and Tobin(1995) Plant MoL Biol. 27:809-813), that of the cabl40 gene ofArabidopsis, the use of the promoter (as a 1.3 kb fragment) and just 14bp of transcribed region fused to a completely unrelated gene wassufficient to result in cosuppression of the endogenous cabl40 gene aswell as the introduced chimeric gene. This result is unusual andapparantly quite unpredictable, as numerous other promoter-leader (the5′ untranslated leader being defined as the region between the start oftranscription and the translation initiation codon) units have been usedto drive chimeric genes successfully. Flavell speculates that some ormany genes (including members of multigene families such as thoseencoding seed proteins) may have evolved so as to avoid the mechanismsof cosuppression, while others have not, providing a potential furtherlevel of regulation as genomes evolve. Thus, the instant observationthat the promoter and leader of the conglycinin gene can be used tosuppress expression of endogenous conglycin3 while the other portion ofthe transgene (beyond the initiation codon) can be used to suppress acompletely unrelated gene is unique.

EXAMPLES

The present invention is further defined by the following examples. Itwill be understood that the examples are given for illustration only andthe present invention is not limited to uses described in the examples.The present invention can be used to generate transgenic soybean plantswith altered levels of various seed storage proteins. From the abovediscussion and the following examples, one skilled in the art canascertain, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. All such modifications are intended tofall within the scope of the intended claims.

Detailed procedures for DNA manipulation, such as use of restrictionendonuelease enzymes, other modifying enzymes, agarose gelelectrophoresis, nucleic acid hybridization, and transformation of E.coli with plasmid DNA are described in Sambrook et al. (1989) MolecularCloning, A Laboratory manual, 2nd ed, Cold Spring Harbor LaboratoryPress (hereinafter “Maniatis”). All restriction enzymes and othermodifying enzymes were obtained from Gibco BRL (Gaithersburg, Mass.).

Example 1

To determine whether the expression of β-conglycinin in developingsoybean cotyledons could be the target of cosuppression, truncated cDNAfragments of the α and α′ subunits of β-conglycinin were prepared usinga reverse transcriptase polymerase chain reaction kit (Geneamp™ RNA PCRKit; Perkin Elmer Cetus). The upper primer, ConS, is homologous tonucleotides 5-19 of the α and α′ subunit cDNA sequences obtained fromthe EMBL/GenBank/DDBJ databases. To aid cloning, additional nucleotideswere added to the 5′ end to code for an Nco I restriction site. Thelower primer, Con 1.4a, is complementary to nucleotides 1370-1354 of SEQID NO:1 and 1472-1456 of SEQ ID NO:2, representing the sequences of theα and α′ cDNAs, respectively. To aid in cloning, additional nucleotideswere added to the 5′ end to introduce a Kpn I restriction site. Thenucleotide sequences of PCR primers ConS and Con1.4a are shown below.

ConS 5′-CGTACCATGGTGAGAGCGCGGTTCC-3′ (SEQ ID NO:4)          Nco I Conl.5′-CGGTACCGAATTGAAGTGTGGTAG-3′ (SEQ ID NO:5) 4a      Kpn I

RNA isolated from developing soybean seeds was reverse-transcribed usingeither the kit-supplied random hexamers, or Con1.4a, following themanufacturer's protocol. The resulting cDNA fragments were amplified inthe PCR (Polymerase Chain Reaction) reaction using a mixture of ConS andCon1.4a. Reactant concentrations were as described in the manufacturer'sprotocols. The following program was used: a) one cycle of 2 minutes at95° C.; b) 35 cycles of: 1.5 minutes at 50° C. (annealing), 5 minutes at70° C. (extension), 1.5 minutes at 95° C. (denaturation), and c) onecycle of 2 minutes at 50° C. followed by 10 minutes at 68° C. Fifteenmicroliters of each of the PCR reaction mixes was analyzed by agarosegel electrophoresis. Reactions resulted in PCR products of the expectedsizes: 1.47 kb for α′ and 1.37 kb for α. The truncated cDNA fragmentsfrom the remainder of the reaction mixes were purified using the Wizard™PCR Preps DNA Purification System kit (Promega).

The purified reaction mix containing the α and α′ fragments, whichbecause of the primers used, included Nco I restriction sites at the 5′ends and Kpn I restriction sites at the 3′ ends, were digested with KpnI and Nco I restriction enzymes. The α cDNA fragment was recoveredfollowing gel electrophoresis, designated as fragment F8, anddirectionally cloned (sense orientation) into pCW109 (FIG. 1) and pML70(FIG. 2) using the Nco I to Kpn I sites present in both plasmids. F8 wasconfirmed as a by PCR using a nested set of primers (Con.09 and Con.8)internal to ConS and Con1.4a, and distinguished from α′ by digestion ofpCW109/F8 plasmid with Hind III, Nco I, Kpn I, and Pst I (α does notcontain a Pst I site whereas α does).

(SEQ ID NO:6) Con.09 5′-TCGTCCATGGAGCGCGGTTCCCATTAC-3′ (SEQ ID NO:7)Con.8 5′-TCTCGGTCGTCGTTGTT-3′

The transcriptional unit KTi promoter/truncated a/KTi 3′ end wasreleased from plasmid pML70/F8 by restriction digest with BamHI, gelisolated, and labeled as F11. F11 was then cloned into pKS18HH (FIG. 3)at the BamH I site. pKS18HH is a plasmid construction containing thefollowing genetic elements: (i) T7 promoter/Hygromycin BPhosphotransferase (HPT)/T7 Terminator Sequence; (ii) 35S promoter fromcauliflower mosaic virus (CaMV)/Hygromycin B Phosphotransferase(HPT)/Nopaline Synthase (NOS) 3′ from Agrobacterium tumefaciens T-DNA;and (iii) pSP72 plasmid vector (Promega) with beta-lactamase codingregion removed. One skilled in the art of molecular biology can ligatethe above three components into a single plasmid vector using well knownprotocols (Maniatis).

The Hygromycin B Phosphotransferase (HPT) gene was isolated by PCRamplification from E. coli strain W677 containing a Klebsiella-derivedplasmid pJR225 (Gritz L., and Davies J. (1983) Gene 25:179-188). pKS18HHcontains the CaMV 35S/HPT/NOR cassette for constitutive expression ofthe HPT enzyme in plants, such as soybean. The pKS18HH plasmid alsocontains the T7 promoter/HPT/T7 terminator cassette for expression ofthe HPT enzyme in certain strains of E. coli, such as NovaBlue® (DE3)(Novagen) that are lysogenic for lambda DE3 (which carries the T7 RNAPolymerase gene under lacUV5 control). pKS18HH also contains threeunique restriction endonuclease sites suitable for cloning of genes intothis vector. Thus, the pKS18HH plasmid vector allows the use ofHygromycin B for selection in both E. coli and plants. Confirmation ofinsertion and orientation of the F11 fragment was accomplished bydigestion with HindIII. A clone with the F11 fragment in clockwiseorientation was selected and labeled pJo1 (FIG. 4).

Transformation of Somatic Embryo Cultures

The following stock solutions and media were used for transformation andpropogation of soybean somatic embryos:

Stock Solutions (g/L) Media MS Sulfate 100× stock SB55 (per Liter) MgSO₄7H₂O 37.0 10 mL of each MS stock MnSO₄ H₂O 1.69 1 mL of B5 Vitamin stockZnSO₄ 7H₂O 0.86 0.8 g NH₄NO₃ CuSO₄ 5H₂O 0.0025 3.033 g KNO₃ 1 mL 2,4-D(10 mg/mL stock) MS Halides 100× stock CaCl₂ 2H₂O 44.0 0.667 gasparagine KI 0.083 pH 5.7 CoCl₂ 6H₂O 0.00125 KH₂PO₄ 17.0 SB103 (perLiter) H₃BO₃ 0.62 1 pk. Murashige & Skoog salt mixture (Gibco BRL)Na₂MoO₄ 2H₂O 0.025 60 g maltose Na₂EDTA 3.724 2 g gelrite FeSO₄ 7H₂O2.784 pH 5.7 (For SB103 plus charcoal, add 5 g charcoal) B5 Vitaminstock SB148 (per Liter) myo-inositol 100.0 1 pk. Murashige & Skoog saltmixture (Gibco BRL) nicotinic acid 1.0 60 g maltose pyridoxine HCl 1.0 1mL B5 vitamin stock thiamine 10.0 7 g agarose pH 5.7

Soybean embryonic suspension cultures were maintained in 35 mL liquidmedia (SB55) on a rotary shaker (150 rpm) at 28° C. with a mix offluorescent and incandescent lights providing a 16/8 h day/nightschedule. Cultures were subcultured every 2 to 3 weeks by inoculatingapproximately 35 mg of tissue into 35 mL of liquid media.

Soybean embryonic suspension cultures were transformed with pJo1 by themethod of particle gun bombardment (see Klein et al. (1987) Nature327:70). A DuPont Biolistic™ PDS1000/He instrument was used for thesetransformations.

Five μL of pJo1 plasmid DNA (1 μg/μL), 50 μL CaCl₂ (2.5 M), and 20 μLspermidine (0.1 M) were added to 50 μL of a 60 mg/mL 1 mm gold particlesuspension. The particle preparation was agitated for 3 minutes, spun ina microfuge for 10 seconds and the supernatant removed. The DNA-coatedparticles were then washed once with 400 μL 70% ethanol and resuspendedin 40 μL of anhydrous ethanol. The DNA/particle suspension was sonicatedthree times for 1 second each. Five μL of the DNA-coated gold particleswere then loaded on each macro carrier disk.

Approximately 300 to 400 mg of two week old suspension culture wasplaced in an empty 60 mm×15 mm petri dish and the residual liquidremoved from the tissue by pipette. The tissue was placed about 3.5inches away from the retaining screen and bombarded twice. Membranerupture pressure was set at 1000 psi and the chamber was evacuated to−28 inches of Hg. Two plates were bombarded per construct perexperiment. Following bombardment, the tissue was divided in half andplaced back into liquid media and cultured as described above.

Fifteen days after bombardment, the liquid media was exchanged withfresh SB55 containing 50 mg/mL hygromycin. The selective media wasrefreshed weekly. Six weeks after bombardment, green, transformed tissuewas isolated and inoculated into flasks to generate new transformedembryonic suspension cultures.

Transformed embryonic clusters were removed from liquid culture mediaand placed on a solid agar media, SB103, plus 0.5% charcoal to beginmaturation. After 1 week, embryos were transferred to SB103 media minuscharcoal. After 3 weeks on SB103 media, maturing embryos were separatedand placed onto SB148 media. Conditions during embryo maturation were26° C., with a mix of fluorescnt and incandescent lights providing a16/8 h day/night schedule. After 6 weeks on SB148 media, embryos wereanalyzed for the expression of the β-conglycinin subunit proteins. Eachembryonic cluster gave rise to 5 to 20 somatic embryos.

Analysis of Transformed Somatic Embryos

Initial experiments were performed to determine when the α, α′ and βsubunits of β-conglycinin could be visualized during somatic embryomaturation by SDS-PAGE gel electrophoresis. Cotyledons ofnon-transformed embryos (generated as above, except they did not undergobombardment) were dissected from embryos at 6, 8, 10, and 12 weeks afterinitiating maturation and kept frozen at −80° C. until analyzed.Cotyledonary tissue was weighed, 10 μL/mg tissue of extraction bufferwas added, and the tissue ground in a Pellet Pestle Disposable Mixer(Kimble/Kontes). Extraction buffer consisted of 50 mM Tris-HCl (pH 7.5),10 mM β-mercaptoetianol (BME), and 0.1% SDS. The samples were thenmicrofaiged at 12,000 rpm for 10 minutes and supernatant remove to a newmicrofuge tube by pipette. Extracts were kept frozen at −20° C. untilused.

For SDS-PAGE analysis, 8 μL of (2×) loading buffer was added to 8 μL ofsample extract. The (2×) loading buffer consisted of 100 mM Tris-HCl (pH7.5), 4% SDS, 0.2% bromophenol blue, 15% glycerol, and 200 mM βME. Themixture was heated at 95° C. for 4 minutes. Sample mixes were thenmicrofuged (12,000 rpm for 20 seconds) and loaded onto a 10% precastReady Gel™ (Bio-Rad) that was assembled into a mini-Protein IIElectrophoresis Cell (Bio-Rad). Bio-Rad Tris/Glycine/SDS Buffer was usedas the running buffer and voltage was a constant 125V. In addition tosample extracts, each gel contained one lane with a molecular weightstandard (Bio-rad SDS-PAGE standard, low range) and one lane with totalsoybean seed protein extracted from commercial defatted soy flour. Uponcompletion, the gels were stained with Coomassie Brilliant Blue anddestained (Maniatis) in order to visualize proteins. Gels werephotographed, placed in a sealed bag with water, and stored in therefrigerator. Results indicated that the α, α′ and β subunits ofβ-conglycinin were detectable in the cotyledons of somatic embryosbetween 8 and 10 weeks after the start of maturation.

Analysis of transformed embryos was carried out at 10 weeks after thestart of maturation using the methods described above. Two embryos perclone were analyzed initially. Additional embryos were analyzed ifsuppression of the β-conglycinin subunits was observed in the twoembryos. Table 1 presents the results of this analysis, wherein thepresence or absence of each β-conglycinin subunit is indicated by a (+)or (−), respectively.

TABLE 1 Clone Embryo α α′ β Jo1-1 1 − − + 2 − − + 3 + + + 4 − − +5 + + + Jo1-2 1 + + + 2 + + + Jo1-3 1 + + + 2 + + + Jo1-4 1 − − − 2 − −− 3 − − − 4 + + + 5 − − − Jo1-5b 1 + + + 2 + + + Jo1-5c 1 − − + 2 − − +Jo1-5d 1 + + + 2 + + + Jo1-6a 1 − − + 2 − − + 3 − − + 4 − − + 5 + + +Jo1-6b 1 + + + 2 + + + Jo1-6c 1 + + + Jo1-6d 1 + + + 2 + + + Jo1-6d1 + + + 2 + + + Jo1-6e 1 + + + 2 + + + Jo1-7a 1 − − + 2 + + + Jo1-7b 1 −− + Jo1-8a 1 + + + Jo1-8b 1 + + + 2 + + + Jo1-9a 1 + + + 2 + + + Jo1-9b1 + + + 2 − − + Jo1-9c 1 + + + Jo1-10 1 − − + 2 + + +

Seven transgenic clones gave rise to embryos in which the expression aand α′ was suppressed. In addition, one clone (Jo1-4) gave rise toembryos in which all three β-conglycinin subunits were suppressed. Thisresult is surprising as the truncated α transgene sequence overlaps withonly a 0.75 kb portion of the total 1.32 kb β subunit cDNA. Overall,there is only 52% similarity between the truncated α transgene and the βsubunit cDNA. With the knowledge at hand, the truncated α transgenewould not be considered to possess sufficient similarity of structure to“cosuppress” the β subunit of the β-conglycinin gene.

An example of an SDS-PAGE analysis is shown in FIG. 5. Lanes 1-3 areextracts of three cotyledons dissected from embryos generated from cloneJo1-1. Lanes 4 and 5 are protein molecular weight standards and soyprotein standard derived from seed, respectively. Lanes 6-8 are extractsof cotyledons dissected from embryos generated from clone Jo1-4. Theprotein pattern in lane 2 is an example of embryos in which both α andα′ are co-suppressed. The protein patterns in lanes 6 and 8 are examplesof embryos where all the subunits comprising β-conglycinin aresuppressed.

Example 2

To determine if expression of β-conglycinin could be suppressed indeveloping cotyledons by cosuppression using the β-conglycinin promoterregion, a plasmid, designated pBS43, containing a Glycine max microsomaldelta-12 desaturase cDNA (GmFad2-1) sequence (Heppard et al., (1996)Plant Physiol. 110:311-319; GenBank Acc. No. L43920) under control ofthe soybean β-conglycinin promoter (Beachy et al., (1985) EMBO J.4:3047-3053), was constructed. The construction of this vector wasfacilitated by the use of the following plasmids: pMH40, pCST2 andpBS13. The plasmid constructions detailed below are described in part inUnited States Patent Application No. U.S. Ser. No. 08/262,401 and WorldPatent Publication No. WO94/11516, both of which are incorporated hereinby reference.

The pMH40 vector was derived from plasmid pGEM9z, a commerciallyavailable cloning vector (Promega Biotech) by the insertion a 1.4 kb 35Spromoter region from CaMV (Odell et al. (1985) Nature 303:810-812;Harpster et al. (1988) Mol. Gen. Genet. 212:182-190) coupled to theβ-glucuronidase gene from E. coli. This was a 1.85 kb fragment encodingthe enzyme β-glucuronidase (Jefferson et al. (1986) PNAS USA83:8447-8451) and a 0.3 kb DNA fragment containing the transcriptionterminator from the nopaline synthase gene of the Ti-plasmid ofAgrobacterium tumefaciens (Fraley et al. (1983) PNAS USA 80:4803-4807).

The vector pCST2 was derived from vectors pML18 and pCW109A. The plasmidpCW109A contains the soybean β-conglycinin promoter sequence and thephaseolin 3′ untranslated region and is a modified version of vectorpCW109 which was derived from the commercially available plasmid pUC18(Gibco-BRL). The vector pCW109 was made by inserting into the Hind IIIsite of the cloning vector pUC18 a 555 bp 5′ non-coding region(containing the promoter region) of the β-conglycinin gene followed bythe multiple cloning sequence containing the restriction endonucleasesites for Nco I, Sma I, Kpn I and Xba I, then 1174 bp of the common beanphaseolin 3′ untranslated region into the Hind III site. Theβ-conglycinin promoter region used is an allele of the publishedβ-conglycinin gene (Doyle et al., (1986) J. Biol. Chem. 261:9228-9238)due to differences at 27 nucleotide positions. Further sequencedescription of this gene may be found World Patent PublicationWO91/13993.

To facilitate use in antisense constructions, the Nco I site andpotential translation start site in the plasmid pCW109 was destroyed bydigestion with Nco I, mung bean exonuclease digestion and religation ofthe blunt site to give the modified plasmid pCW109A.

The vector pML18 consists of the non-tissue specific and constitutivecauliflower mosaic virus (35S) promoter (Odell et al., (1985) Nature313:810-812; Hull et al., (1987) Virology 86:482-493), drivingexpression of the neomycin phosphotransferase gene (Beck et al. (1982)Gene 19:327-336) followed by the 3′ end of the nopaline synthase geneincluding nucleotides 848 to 1550 (Depicker et al. (1982) J. Appl.Genet. 1:561-574). This transcriptional unit was inserted into thecommercial cloning vector pGEM9z (Gibco-BRL) and is flanked at the 5′end of the 35S promoter by the restriction sites Sal I, Xba I, Bam HIand Sma I, in that order. An additional Sal I site is present at the 3′end of the NOS 3′ sequence and the Xba I, Bam HI and Sal I sites areunique. The plasmid pML18 was digested with Xba I, the singled strandedends were filled-in using the Klenow fragment of DNA polymerase I, andthe product was ligated in order to remove the Xba I site. The resultingplasmid was designated pBS16.

The plasmid pCW109A was digested with Hind III and the resulting 1.84 kbfragment, which contained the β-conglycinin/antisense delta-12desaturase cDNA/phaseolin 3′ untranslated region, was gel isolated. This1.84 kb fragment was ligated into the Hind III site of pBS16. A plasmidcontaining the insert in the desired orientation yielded a 3.53 kb and4.41 kb fragment when digested with Kpn I and this plasmid wasdesignated pCST2.

The vector pBS13 was used as the source of the GmFad2-1 cDNA, whichencodes the soybean microsomal delta2-desaturase and possesses thesequence as disclosed in GenBank Acc. No. L43920. The vector pBS13 wasderived from the vector pML70 (FIG. 1), which contains the KTi3 promoterand the KTi3 3′ untranslated region and was derived from thecommercially available vector pTZ18R (Pharmacia) via the intermediateplasmids pML51, pML55, pML64 and pML65. A 2.4 kb Bst BI/Eco RI fragmentof the complete soybean KTi3 gene (Jofuku and Goldberg (1989) Plant Cell1:1079-1093), which contains all 2039 nucleotides of the 5′ untranslatedregion and 390 bases of the coding sequence of the KTi3 gene ending atthe Eco RI site corresponding to bases 755 to 761 of the sequencedescribed in Jofuku (supra), was ligated into the Acc I/Eco RI sites ofpTZ18R to create the plasmid pML51. To destroy an Nco I site in themiddle of the 5′ untranslated region of the KTi3 insert, plasmid pML51was cut with Nco I, the singled stranded ends were filled-in using theKlenow fragment of DNA polymerase I, and the product was religatedresulting in the plasmid pML55. The plasmid pML55 was partially digestedwith Xmn I/Eco RI to release a 0.42 kb fragment, corresponding to bases732 to 755 of the above cited sequence, which was discarded. A syntheticXmn I/Eco RI linker containing an Nco I site, was constructed by makinga dimer of complementary synthetic oligonucleotides consisting of thecoding sequence for an Xmn I site (5′-TCTTCC-3′) and an Nco I site(5′-CCATGGG-3′) followed directly by part of an Eco RI site(5′-GAAGG-3′). The Xmn I and Nco I/Eco RI sites were linked by a shortintervening sequence (5′-ATAGCCCCCCAA-3′)(Seq ID No:22). This syntheticlinker was ligated into the Xmn I/Eco RI sites of the 4.94 kb fragmentto create the plasmid pML64. The 3′ untranslated region of the KTi3 genewas amplified from the sequence described in Jofuku (supra) by standardPCR protocols (Perkin Elmer Cetus, GeneAmp PCR kit) using the primersML51 and ML52. Primer ML51 contained the 20 nucleotides corresponding tobases 1072 to 1091 of the above cited sequence with the addition ofnucleotides corresponding to Eco RV (5-′GATATC-3′), Nco I(5′-CCATGG-3′), Xba I (5′-TCTAGA-3′), Sma I (5′-CCCGGG-3′) and Kpn I(5′-GGTACC-3′) sites at the 5′ end of the primer. Primer ML52 containedto the exact complement of the nucleotides corresponding to bases 1242to 1259 of the above cited sequence with the addition of nucleotidescorresponding to Sma I (5′-CCCGGG-3′), Eco RI (5′-GAATTC-3′), Bam HI(5′-GGATCC-3′) and Sal I (5′-GTCGAC-3′) sites at the 5′ end of theprimer. The PCR-amplified 3′ end of the KTi3 gene was ligated into theNco I/Eco RI sites of pML64 to create the plasmid pML65. A syntheticmultiple cloning site linker was constructed by making a dimer ofcomplementary synthetic oligonucleotides consisting of the codingsequence for Pst I (5′-CTGCA-3′), Sal I (5′-GTCGAC-3′), Bam HI(5′-GGATCC-3′) and Pst I (5′-CTGCA-3′) sites. The linker was ligatedinto the Pst I site (directly 5′ to the KTi3 promoter region) of pML65to create the plasmid pML70.

The 1.46 kb Sma I/Kpn I fragment from soybean delta-12 desaturase cDNA,GmFad2-1 (GenBank Acc. No. L43920), was ligated into the correspondingsites in pML70 resulting in the plasmid pBS10. The desaturase cDNAfragment was in the reverse (antisense) orientation with respect to theKTi3 promoter in pBS10. The plasmid pBS10 was digested with Bam HI and a3.47 kb fragment, representing the KTi3 promoter/antisense desaturasecDNA/KTi3 3′ end transcriptional unit was isolated by agarose gelelectrophoresis. The vector pML18 consists of the non-tissue specificand constitutive cauliflower mosaic virus (35S) promoter (Odell et al.,(1985) Nature 313:810-812; Hull et al., (1987) Virology 86:482-493),driving expression of the neomycin phosphotransferase gene (Beck et al.(1982) Gene 19:327-336) followed by the 3′ end of the nopaline synthasegene including nucleotides 848 to 1550 (Depicker et al. (1982) J. Appl.Genet. 1:561-574). This transcriptional unit was inserted into thecommercial cloning vector pGEM9z (Gibco-BRL) and is flanked at the 5′end of the 35S promoter by the restriction sites Sal I, Xba I, Bam HIand Sma I in that order. An additional Sal I site is present at the 3′end of the NOS 3′ sequence and the Xba I, Bam HI and Sal I sites areunique. The 3.47 kb transcriptional unit released from pBS10 was ligatedinto the Bam HI site of the vector pML18. When the resulting plasmidswere digested with Sma I and Kpn I, plasmids containing inserts in thedesired orientation yielded 3 fragments of 5.74, 2.69 and 1.46 kb. Aplasmid with the transcriptional unit in the correct orientation wasselected and was designated pBS13.

The 1.46 kb XbaI/EcoRV fragment from pBS13 (described above) wasdirectionally cloned into the SmaI/XbaI site of vector pCST2 (describedabove) to yield a plasmid designated pBS39. The 3.3 kb HindIII fragmentof plasmid pBS39 was cloned into the HindIII site of plasmid pMH40(described above) to give the plant expression vector pBS43 (FIG. 6).

Transformation of Soybeans with Vector pBS43 and Identification of aTransgenic “Transwitch” Line

The vector pBS43 was transformed into soybean meristems using the methodof particle bombardment of soybean meristems (Christou et al (1990)Trends Biotechnol. 8:145-151). Seeds of transformed plants (i.e., fromplants which had been identified as positive for GUS activity) werescreened for fatty acid composition. Fatty acid methyl esters wereprepared from hexane extracts of small (approx. 10 mg) seed chips(Browse et al (1986) Anal. Biochem. 152:141-145). Seed chips from tendifferent transgenic lines were analysed and some of the R1 seeds fromone of these lines, designated 260-05, had a total oleic acid content of80-85% compared with about 20% in control seeds. This phenotype iscaused by the cosuppression of the endogenous Fad 2-1 gene and is theresult of the insertion of two copies of pBS43 into a locus of thesoybean genome designated the “Transwitch locus” (Kinney, A. J. (1995)in “Induced Mutations and Molecular Techniques for Crop Improvement”,International Atomic Energy Agency, Vienna). High oleic acid R1 seedsfrom line 260-05, which contained the Transwitch locus, were selfed andR2 seeds which were homozygous for the Transwitch locus were selected.Two of these R2 homozygous seeds (G94-1, G94-19) and seeds derived fromfurther generations of G94-1 and G94-19 (R3, R4, R5), were selected forfurther analysis.

R5 seeds of G94-1 and G94-19 plants grown in both Iowa and Puerto Ricowere ground into a powder and approximately 1 g extracted with 5 mL ofhexane. After centrifugation, the hexane was poured off and the flakesallowed to air dry. Approximately 10 mg of defatted powder was extractedas described above and analyzed by SDS-PAGE. In both transgenic linesderived from both locations, the expression of the α′ and a subunits ofβ-conglycinin were suppressed relative to control soybean lines and astandard soy flour (FIG. 7).

Example 3

To test if β-conglycinin expression could be suppressed using antisensetechnology, full length cDNAs of α and α′ were made using reversetranscriptase polymerase chain reaction as described above. The upperprimer, ConSa, is homologous to region 4-19 of both α and α′ cDNAsequences with additional nucleotides added to the 5′ end to code for aKpn I restriction site. The lower primer used, Con1.9a, is homologous toregions 1818-1801 of SEQ ID NO:1, representing the α isoform, and1920-1903 of SEQ ID NO:2, representing the α′ isoform, respectively. Toaid in subsequent cloning steps, additional nucleotides were added tothe 5′ end to code for an Nco I restriction site.

(SEQ ID NO:8) ConSa 5′-ACGGTACCGATGAGAGCGCGGTTCC-3′        Kpn I (SEQ IDNO:9) Conl.9a 5′-AACCCATGGTCAGTAAAAAGCCCTCAA-3′         NcoI

Reverse transcription and subsequent PCR reaction were carried out asdescribed above. RNA isolated from developing soybean seeds wasreverse-transcribed using either random hexamers or Con1.9a ( method asdetailed above). The cDNA was amplified in a PCR reaction using ConSaand Con1.9a using the protocol detailed above. Fifteen microliters ofthe PCR reaction mixes were analyzed by agarose gel electrophoresis. A1.8 kb band, the expected molecular weight for α, was observed. Theremaining reaction mixes were purified using Wizard™ PCR Preps DNAPurification System kit (Promega). The α cDNA, which because of theprimers used included a Kpn I site on the 5′ end and an Nco I site onthe 3′ end, was digested with Nco I and Kpn I restriction enzymes. Theresulting αcDNA was gel isolated, labeled as F10, and directionallycloned (antisense orientation) into pCW109 using the Nco I and Kpn Isites present in the plasmid. F10 was confined as α by PCR using nestedprimers (upper: Con.09 (SEQ ID NO:6); lower: Con1.4a (SEQ ID NO:5) andCon1.0 (SEQ ID NO:10)).

Conl.0 5′-CGGGTATGGCGAGTGTT-3′ (SEQ ID NO:10)

The transcriptional unit β-conglycinin promoter/α cDNAantisense/phaseolin 3′ end was released from pCW109/F10 by partialdigest with Hind III. Conditions of the partial digest were such that 6fragments were produced (5.1 kb, 3.8 kb, 3.6 kb, 2.6 kb, 2.4 kb, and 1.2kb). The 3.6 kb fragment containing the the transcriptional unit was gelisolated and labeled F14. F14 was then cloned into the Hind III site ofpKS 1 8HH. After confirming insertion by digestion of plasmid DNApreparations made from tansformed cells with Hind III, the plasmid DNAfrom positive cultures was digested with Kpn I to ensure that theycontained the 3.6 kb F14 fragment and not the 3.8 kb fragment from thepartial digest of pCW109/F10 with Hind III. F14 contains a Kpn I site,while the 3.8 kb fragment does not. Upon confirmation, pKS18HH/F14 waslabeled pJo3 (FIG. 8). Soybean embryonic suspension cultures weretransformed with pJo3 as detailed above. Transformation resulted in 5transformed clones; upon maturation each clone gave rise to 4 to 8somatic embryos.

Protein extracts of transformed somatic embryos were analyzed bySDS-PAGE as previously detailed. Results are presented in Table 2. Thetransgenic clones all gave rise to at least one somatic embryo in whichthe expression of both α and α′ was suppressed.

TABLE 2 Clone Embryo α α′ β Jo3-1 1 − − + 2 + + + Jo3-2 1 − − + 2 − − +Jo3-2b 1 − − + 2 − − + Jo3-3 1 − − + 2 − − + Jo3-4 1 − − + 2 − − +

Example 4

There are five non allelic genes that code for the glycinin subunits.

Sequencing genomic clones and cDNA's have lead to a division of thesubunit genes into two groups based on sequence similarity. Group Iconsists of Gyl (SEQ ID NO:11), Gy2 (SEQ ID NO:12) and Gy3 (SEQ IDNO:13), whereas group II consists of Gy4 (SEQ ID NO:14) and GyS (SEQ IDNO:15). There is greater than 85% similarity between genes within agroup, but only 42% to 46% similarity between genes of different groups.To determine whether expression of glycinin can be suppressed indeveloping cotyledons by employing co-suppression technology, cDNA's ofGroup I and Group II were prepared using reverse transcriptasepolymerase chain reaction as described above.

The upper primer used for Group I reactions (G1-1) is homologus toregions 1-19 for all Group I cDNA's. Two lower primers were used:G1-1039, which is homologous with regions 1038-1022 of Gy1, 1008-992 ofGy2, and 996-980 of Gy3; or G1-1475, which is homologus to regions1475-1460 of Gy1, 1445-1430 of Gy2 and 1433-1418 of Gy3. To aid infuture cloning, all primers contained additional nucleotides that codedfor a Not I restriction site at their 5′ end.

(SEQ ID NO:16) G1-1 5′-GCGGCCGCATGGCCAAGCTAGTTTTTT-3′        Not I (SEQID NO:17) G1-1039 5′-GCGGCCGCTGGTGGCGTTTGTGA-3′        Not I (SEQ IDNO:18) G1-1475 5′-GCGGCCGCTCTTCTGAGACTCCT-3′ (SEQ ID NO:18)        Not I

RNA isolated from developing soybean seeds was reverse-transcribed usingeither random hexamers, or G1-1475 or G1-1039 as the lower primer in thereactions. cDNA fragments were amplified using a mixture of G1-1 witheither G1-1039 or G1-1475. Fifteen microliters of the PCR reaction mixeswere analyzed by agarose gel electrophoresis. PCR reactions resulted inproducts of the expected molecular weight, approximately 1 kb and1.4-1.5 kb for primer sets G1-1/G1-1039 and G1-1/G1-1475, respectively.cDNA fragments from the remainder of the reaction mixes were purifiedusing the Wizard™ PCR Preps DNA Purification System kit (Promega).Purified cDNA's were then digested with Not I and isolated by agarosegel purification.

The upper primer used for RT-PCR reactions of Group II (G4-7) ishomologus to regions 7-22 for both cDNA's of Group II. Two lower primerswere used: G4-1251 which is homologus with regions 1251-1234 of Gy4 and1153-1135 of GyS; or G4-1670 which is homologus to regions 1668-1653 ofGy4. There is no similar region in Gy5. To aid in future cloning allprimers contained additional nucleotides that coded for a Not Irestriction site at their 5′ end.

(SEQ ID NO:19) G4-7 5′-GCGGCCGCATGCCCTTCACTCTCT-3′        Not I (SEQ IDNO:20) G4-1251 5′-GCGGCCGCTGGGAGGGTGAGGCTGTT-3′        Not I (SEQ IDNO:21) G4-1670 5′-GCGGCCGCTGAGCCTTGTTGAGAC-3′        Not I

RNA isolated from developing soybean seeds was reverse-transcribed usingeither random hexamers, or G4-1251 or G4-1670 as the lower primer in thereactions. cDNA fragments were amplified using a mixture of G4-7 witheither G4-1251 or G4-1670. Fifteen microliters of the PCR reaction mixeswere analyzed by agarose gel electrophoresis. PCR reactions resulted inproducts of the expected molecular weight, approximately 1.25 kb and 1.7kb for primer sets G4-7/G4-1251 and G4-7/G4-16.70, respectively. cDNAfragments from the remainder of the reaction mixes were purified usingthe Wizard™ PCR Preps DNA Purification System kit (Promega). PurifiedcDNA's were then digested with Not I and isolated from gels.

The isolated group I cDNAs are cloned into pRB20 (FIG. 9) at the Not Isite (sense orientation). After partial restriction digest with Not Iand isolation of the single cut pRB20/group I linear fragments, group IIcDNA are added to create final transcriptional units β-conglycininpromoter/group I cDNA (sense orientation)/phaseolin 3′ end andβ-conglycinin promoter/group II cDNA (sense orientation)/phaseolin 3′end. The resulting plasmids are then used to transform somatic embryonicsuspension cultures using the method detailed above.

22 1818 base pairs nucleic acid single linear cDNA 1 ATGATGAGAGCACGGTTCCC ATTACTGTTG CTGGGACTTG TTTTCCTGGC TTCAGTTTCT 60 GTCTCATTTGGCATTGCTTA CTGGGAAAAA GAGAACCCCA AACACAACAA GTGTCTCCAG 120 AGTTGCAATAGCGAGAGAGA CTCGTACAGG AACCAAGCAT GCCACGCTCG TTGCAACCTC 180 CTTAAGGTGGAGAAAGAAGA ATGTGAAGAA GGTGAAATTC CACGACCACG ACCACGACCA 240 CAACACCCGGAGAGGGAACC TCAGCAACCC GGTGAGAAGG AGGAAGACGA AGATGAGCAA 300 CCACGTCCAATCCCATTCCC ACGCCCACAA CCTCGTCAAG AAGAAGAGCA CGAGCAGAGA 360 GAGGAACAGGAATGGCCTCG CAAGGAGGAA AAACGCGGAG AAAAGGGAAG TGAAGAGGAA 420 GATGAGGATGAGGATGAGGA ACAAGATGAA CGTCAATTCC CATTCCCACG CCCACCTCAT 480 CAGAAGGAAGAGCGAAACGA AGAGGAAGAT GAGGATGAGG AGCAGCAGCG AGAGAGCGAA 540 GAAAGTGAAGATTCTGAGTT ACGAAGACAT AAGAATAAGA ACCCTTTTCT CTTCGGCTCT 600 AACAGGTTCGAAACTCTCTT CAAAAACCAA TATGGTCGCA TTCGCGTCCT CCAGAGGTTC 660 AACCAACGCTCCCCACAACT TCAGAATCTC CGAGACTACC GCATTTTGGA GTTCAACTCC 720 AAACCCAACACCCTCCTTCT CCCCAACCAT GCTGACGCTG ATTACCTCAT CGTTATCCTT 780 AACGGGACTGCCATTCTTTC CTTGGTGAAC AACGACGACA GAGACTCCTA CAGACTTCAA 840 TCTGGTGATGCCCTGAGAGT CCCCTCAGGA ACCACATACT ATGTGGTCAA CCCTGACAAC 900 AACGAAAATCTCAGATTAAT AACACTCGCC ATACCCGTTA ACAAGCCTGG TAGATTTGAG 960 AGTTTCTTCCTATCTAGCAC TGAAGCTCAA CAATCCTACT TGCAAGGATT CAGCAGGAAC 1020 ATTTTAGAGGCCTCCTACGA TACCAAATTC GAGGAGATAA ACAAGGTTCT GTTTAGTAGA 1080 GAGGAAGGGCAGCAGCAAGG GGAGCAGAGG CTGCAAGAGA GCGTGATTGT GGAAATCTCG 1140 AAGGAACAGATTCGGGCACT GAGCAAACGT GCCAAATCTA GTTCAAGGAA AACCATTTCT 1200 TCTGAAGATAAACCTTTTAA CTTGAGAAGC CGCGACCCCA TCTACTCCAA CAAGCTTGGC 1260 AAGTTCTTTGAGATCACCCC AGAGAAAAAC CCCCAGCTTC GGGACTTGGA TATCTTCCTC 1320 AGTATTGTGGATATGAACGA GGGAGCTCTT CTTCTACCAC ACTTCAATTC AAAGGCGATA 1380 GTGATACTGGTAATTAATGA AGGAGATGCA AACATTGAAC TTGTTGGCCT AAAAGAACAA 1440 CAACAGGAGCAGCAACAGGA AGAGCAACCT TTGGAAGTGC GGAAATATAG AGCCGAATTG 1500 TCTGAACAAGATATATTTGT AATCCCAGCA GGTTATCCAG TTGTGGTCAA CGCTACCTCA 1560 AATCTGAATTTCTTTGCTAT TGGTATTAAT GCCGAGAACA ACCAGAGGAA CTTCCTCGCA 1620 GGTTCGCAAGACAATGTGAT AAGCCAGATA CCTAGTCAAG TGCAGGAGCT TGCATTCCCT 1680 GGGTCTGCACAAGCTGTTGA GAAGCTATTA AAGAACCAAA GAGAATCCTA CTTTGTGGAT 1740 GCTCAGCCTAAGAAGAAAGA GGAGGGGAAT AAGGGAAGAA AGGGTCCTTT GTCTTCAATT 1800 TTGAGGGCTTTTTACTGA 1818 1920 base pairs nucleic acid single linear cDNA 2ATGATGAGAG CGCGGTTCCC ATTACTGTTG CTGGGAGTTG TTTTCCTAGC ATCAGTTTCT 60GTCTCATTTG GCATTGCGTA TTGGGAAAAG CAGAACCCCA GTCACAACAA GTGCCTCCGA 120AGTTGCAATA GCGAGAAAGA CTCCTACAGG AACCAAGCAT GCCACGCTCG TTGCAACCTC 180CTTAAGGTGG AGGAAGAAGA AGAATGCGAA GAAGGTCAAA TTCCACGACC ACGACCACAA 240CACCCGGAGA GGGAACGTCA GCAACACGGT GAGAAGGAGG AAGACGAAGG TGAGCAGCCA 300CGTCCATTCC CATTCCCACG CCCACGCCAA CCTCATCAAG AGGAAGAGCA CGAGCAGAAG 360GAGGAACACG AATGGCATCG CAAGGAGGAA AAACACGGAG GAAAGGGAAG TGAAGAGGAA 420CAAGATGAAC GTGAACACCC ACGCCCACAC CAACCTCATC AAAAGGAAGA GGAAAAGCAC 480GAATGGCAAC ACAAGCAGGA AAAGCACCAA GGAAAGGAAA GTGAAGAAGA AGAAGAAGAC 540CAAGACGAGG ATGAGGAGCA AGACAAAGAG AGCCAAGAAA GTGAAGGTTC TGAGTCTCAA 600AGAGAACCAC GAAGACATAA GAATAAGAAC CCTTTTCACT TCAACTCTAA AAGGTTCCAA 660ACTCTCTTCA AAAACCAATA TGGCCACGTT CGCGTCCTCC AGAGGTTCAA CAAACGCTCC 720CAACAGCTTC AGAATCTCCG AGACTACCGC ATTTTGGAGT TCAACTCCAA ACCCAACACC 780CTTCTTCTCC CCCACCATGC TGACGCTGAT TACCTCATCG TTATCCTTAA CGGGACTGCC 840ATTCTTACCT TGGTGAACAA CGACGACCGA GACTCTTACA ACCTTCAATC TGGCGATGCC 900CTAAGAGTCC CTGCAGGAAC CACATTCTAT GTGGTTAACC CTGACAACGA CGAGAATCTC 960AGAATGATAG CAGGAACCAC ATTCTATGTG GTTAACCCTG ACAACGACGA GAATCTCAGA 1020ATGATAACAC TCGCCATACC CGTTAACAAA CCCGGTAGAT TTGAGAGTTT CTTCCTATCT 1080AGCACTCAAG CTCAACAGTC CTACTTGCAA GGGTTCAGCA AGAATATTCT AGAGGCCTCA 1140TACGACACCA AATTCGAGGA GATAAACAAG GTTCTGTTTG GTAGAGAGGA GGGGCAGCAA 1200CAAGGGGAGG AGAGGCTGCA AGAGAGTGTG ATTGTGGAAA TCTCAAAGAA ACAAATTCGG 1260GAACTGAGCA AACATGCCAA ATCTAGTTCA AGGAAAACCA TTTCTTCTGA AGATAAACCT 1320TTCAACTTGG GAAGCCGCGA CCCCATCTAT TCCAACAAGC TTGGCAAGTT GTTTGAGATT 1380ACCCAGAGAA ACCCTCAGCT TCGGGACTTG GATGTCTTCC TCAGTGTTGT GGATATGAAC 1440GAGGGAGCTC TTTTTCTACC ACACTTCAAT TCAAAGGCCA TAGTGGTACT AGTGATTAAT 1500GAAGGAGAAG CAAACATTGA ACTTGTTGGC ATTAAAGAAC AACAACAGAG GCAGCAACAG 1560GAAGAGCAAC CTTTGGAAGT GCGGAAATAT AGAGCTGAAT TGTCTGAACA AGATATATTT 1620GTAATCCCAG CAGGTTATCC AGTTATGGTC AACGCTACCT CAGATCTGAA TTTCTTTGCT 1680TTTGGTATCA ATGCCGAGAA CAACCAGAGG AACTTCCTTG CAGGTTCGAA AGACAATGTG 1740ATAAGCCAGA TACCTAGTCA AGTGCAGGAG CTTGCGTTCC CTAGGTCTGC AAAAGATATT 1800GAGAACCTAA TAAAGAGCCA AAGTGAGTCC TACTTTGTGG ATGCTCAGCC TCAGCAGAAA 1860GAGGAGGGGA ACAAGGGAAG AAAGGGTCCT TTGTCTTCAA TTTTGAGGGC TTTTTACTGA 19201320 base pairs nucleic acid single linear cDNA 3 ATGATGAGAG TGCGGTTTCCTTTGTTGGTG TTGCTGGGAA CTGTTTTCCT GGCATCAGTT 60 TGTGTCTCAT TAAAGGTGAGAGAGGATGAG AATAACCCTT TCTACTTTAG AAGCTCTAAC 120 AGCTTCCAAA CTCTCTTTGAGAACCAAAAC GTTCGCATTC GTCTCCTCCA GAGATTCAAC 180 AAACGCTCCC CACAACTTGAGAACCTTCGA GACTACCGGA TTGTCCAGTT TCAGTCAAAA 240 CCCAACACAA TCCTTCTCCCCCACCATGCT GACGCCGATT TCCTCCTCTT TGTCCTTAGC 300 GGGAGAGCCA TACTTACCTTGGTGAACAAC GACGACAGAG ACTCCTACAA CCTTCACCCT 360 GGCGATGCCC AGAGAATCCCAGCTGGAACC ACTTACTATT TGGTTAACCC TCACGACCAC 420 CAGAATCTCA AAATAATCAAACTTGCCATA CCCGTCAACA AACCTGGCAG ATATGATGAT 480 TTCTTCTTAT CTAGCACTCAAGCCCAACAG TCCTACTTGC AAGGCTTCAG CCATAATATT 540 CTAGAGACCT CCTTCCATAGCGAATTCGAG GAGATAAACA GGGTTTTGTT TGGAGAGGAA 600 GAGGAGCAGA GGCAGCAAGAGGGAGTGATC GTGGAACTCT CAAAGGAACA AATTCGGCAA 660 CTGAGCAGAC GTGCCAAATCTAGTTCAAGG AAAACCATTT CCTCCGAAGA TGAACCATTC 720 AACTTGAGAA GCCGCAACCCCATCTATTCC AACAACTTTG GAAAGTTCTT TGAGATCACC 780 CCTGAGAAAA ACCCACAGCTTCGGGACTTG GATATCTTCC TCAGTTCTGT GGATATCAAC 840 GAAGGAGCTC TTCTTCTACCACACTTCAAT TCAAAGGCCA TAGTGATACT AGTGATTAAT 900 GAAGGAGATG CAAACATTGAACTTGTTGGC ATTAAAGAAC AACAACAGAA GCAGAAACAG 960 GAAGAGGAAC CTTTGGAAGTGCAAAGGTAC AGAGCTGAAT TGTCTGAAGA CGATGTATTT 1020 GTAATTCCAG CAGCTTATCCATTTGTCGTC AACGCTACCT CAAACCTCAA TTTCCTTGCT 1080 TTTGGTATCA ATGCTGAGAACAACCAGAGG AACTTCCTTG CAGGCGAGAA AGACAATGTG 1140 GTAAGGCAGA TAGAAAGACAAGTGCAGGAG CTTGCGTTCC CTGGGTCTGC ACAAGATGTT 1200 GAGAGGCTAT TAAAGAAGCAGAGGGAATCC TACTTTGTTG ATGCTCAGCC TCAGCAGAAG 1260 GAGGAGGGGA GTAAGGGAAGAAAGGGTCCT TTTCCTTCAA TCTTAGGTGC TCTCTACTGA 1320 25 base pairs nucleicacid single linear DNA (genomic) 4 CGTACCATGG TGAGAGCGCG GTTCC 25 24base pairs nucleic acid single linear DNA (genomic) 5 CGGTACCGAATTGAAGTGTG GTAG 24 27 base pairs nucleic acid single linear DNA(genomic) 6 TCGTCCATGG AGCGCGGTTC CCATTAC 27 17 base pairs nucleic acidsingle linear DNA (genomic) 7 TCTCGGTCGT CGTTGTT 17 25 base pairsnucleic acid single linear DNA (genomic) 8 ACGGTACCGA TGAGAGCGCG GTTCC25 27 base pairs nucleic acid single linear DNA (genomic) 9 AACCCATGGTCAGTAAAAAG CCCTCAA 27 17 base pairs nucleic acid single linear DNA(genomic) 10 CGGGTATGGC GAGTGTT 17 1488 base pairs nucleic acid singlelinear cDNA 11 ATGGCCAAGC TAGTTTTTTC CCTTTGTTTT CTGCTTTTCA GTGGCTGCTGCTTCGCTTTC 60 AGTTCCAGAG AGCAGCCTCA GCAAAACGAG TGCCAGATCC AAAAACTCAATGCCCTCAAA 120 CCGGATAACC GTATAGAGTC AGAAGGAGGG CTCATTGAGA CATGGAACCCTAACAACAAG 180 CCATTCCAGT GTGCCGGTGT TGCCCTCTCT CGCTGCACCC TCAACCGCAACGCCCTTCGT 240 AGACCTTCCT ACACCAACGG TCCCCAGGAA ATCTACATCC AACAAGGTAAGGGTATTTTT 300 GGCATGATAT ACCCGGGTTG TCCTAGCACA TTTGAAGAGC CTCAACAACCTCAACAAAGA 360 GGACAAAGCA GCAGACCACA AGACCGTCAC CAGAAGATCT ATAACTTCAGAGAGGGTGAT 420 TTGATCGCAG TGCCTACTGG TGTTGCATGG TGGATGTACA ACAATGAAGACACTCCTGTT 480 GTTGCCGTTT CTATTATTGA CACCAACAGC TTGGAGAACC AGCTCGACCAGATGCCTAGG 540 AGATTCTATC TTGCTGGGAA CCAAGAGCAA GAGTTTCTAA AATATCAGCAAGAGCAAGGA 600 GGTCATCAAA GCCAGAAAGG AAAGCATCAG CAAGAAGAAG AAAACGAAGGAGGCAGCATA 660 TTGAGTGGCT TCACCCTGGA ATTCTTGGAA CATGCATTCA GCGTGGACAAGCAGATAGCG 720 AAAAACCTAC AAGGAGAGAA CGAAGGGGAA GACAAGGGAG CCATTGTGACAGTGAAAGGA 780 GGTCTGAGCG TGATAAAACC ACCCACGGAC GAGCAGCAAC AAAGACCCCAGGAAGAGGAA 840 GAAGAAGAAG AGGATGAGAA GCCACAGTGC AAGGGTAAAG ACAAACACTGCCAACGCCCC 900 CGAGGAAGCC AAAGCAAAAG CAGAAGAAAT GGCATTGACG AGACCATATGCACCATGAGA 960 CTTCGCCACA ACATTGGCCA GACTTCATCA CCTGACATCT ACAACCCTCAAGCCGGTAGC 1020 GTCACAACCG CCACCAGCCT TGACTTCCCA GCCCTCTCGT GGCTCAGACTCAGTGCTGAG 1080 TTTGGATCTC TCCGCAAGAA TGCAATGTTC GTGCCACACT ACAACCTGAACGCGAACAGC 1140 ATAATATACG CATTGAATGG ACGGGCATTG ATACAAGTGG TGAATTGCAACGGTGAGAGA 1200 GTGTTTGATG GAGAGCTGCA AGAGGGACGG GTGCTGATCG TGCCACAAAACTTTGTGGTG 1260 GCTGCAAGAT CACAGAGTGA CAACTTCGAG TATGTGTCAT TCAAGACCAATGATACACCC 1320 ATGATCGGCA CTCTTGCAGG GGCAAACTCA TTGTTGAACG CATTACCAGAGGAAGTGATT 1380 CAGCACACTT TCAACCTAAA AAGCCAGCAG GCCAGGCAGA TAAAGAACAACAACCCTTTC 1440 AAGTTCCTGG TTCCACCTCA GGAGTCTCAG AAGAGAGCTG TGGCTTAG1488 1458 base pairs nucleic acid single linear cDNA 12 ATGGCCAAGCTTGTTCTTTC CCTTTGTTTC CTTCTTTTCA GTGGCTGCTT CGCTCTGAGA 60 GAGCAGGCACAGCAAAATGA GTGCCAGATC CAAAAGCTGA ATGCCCTCAA ACCGGATAAC 120 CGTATAGAGTCGGAAGGTGG GTTCATTGAG ACATGGAACC CTAACAACAA GCCATTCCAG 180 TGTGCCGGTGTTGCCCTCTC TCGCTGCACC CTTAACCGCA ATGCCCTTCG TAGACCTTCC 240 TACACCAACGGTCCCCAGGA AATCTACATA CAACAAGGTA ATGGTATTTT TGGCATGATA 300 TTCCCGGGTTGTCCTAGCAC TTATCAAGAG CCGCAAGAAT CTCAGCAACG AGGACGAAGC 360 CAGAGGCCCCAAGACCGTCA CCAAAAGGTA CATCGCTTCA GAGAGGGTGA TTTGATCGCA 420 GTGCCTACTGGTGTTGCATG GTGGATGTAC AACAATGAAG ACACTCCTGT TGTTGCCGTT 480 TCTATTATTGACACCAACAG CTTGGAGAAC CAGCTCGACC AGATGCCTAG GAGATTCTAT 540 CTTGCTGGGAACCAAGAGCA AGAGTTTCTA AAATATCAGC AGCAGCAGCA AGGAGGTTCC 600 CAAAGCCAGAAAGGAAAGCA ACAAGAAGAA GAAAACGAAG GAAGCAACAT ATTGAGTGGC 660 TTCGCCCCTGAATTCTTGAA AGAAGCGTTC GGCGTGAACA TGCAGATAGT GAGAAACCTA 720 CAAGGTGAGAACGAAGAGGA GGATAGTGGA GCCATTGTGA CAGTGAAAGG AGGTCTAAGA 780 GTCACAGCTCCAGCCATGAG GAAGCCACAG CAAGAAGAAG ATGATGATGA TGAGGAAGAG 840 CAGCCACAGTGCGTGGAGAC AGACAAAGGT TGCCAACGCC AAAGCAAAAG GAGCAGAAAT 900 GGCATTGATGAGACCATTTG CACAATGAGA CTTCGCCAAA ACATTGGTCA GAATTCATCA 960 CCTGACATCTACAACCCTCA AGCTGGTAGC ATCACAACCG CCACCAGCCT TGACTTCCCA 1020 GCCCTCTGGCTTCTCAAACT CAGTGCCCAG TATGGATCAC TCCGCAAGAA TGCTATGTTC 1080 GTGCCACACTACACCCTGAA CGCGAACAGC ATAATATACG CATTGAATGG GCGGGCATTG 1140 GTACAAGTGGTGAATTGCAA TGGTGAGAGA GTGTTTGATG GAGAGCTGCA AGAGGGAGGG 1200 GTGCTGATCGTTCCACAAAA CTTTGCGGTG GCTGCAAAAT CCCAGAGCGA TAACTTTGAG 1260 TATGTGTCATTCAAGACCAA TGATAGACCC TCGATCGGAA ACCTTGCAGG GGCAAACTCA 1320 TTGTTGAACGCATTGCCAGA GGAAGTGATT CAGCACACTT TTAACCTAAA GAGCCAGCAG 1380 GCCAGGCAGGTGAAGAACAA CAACCCTTTC AGCTTCCTTG TTCCACCTCA GGAGTCTCAG 1440 AGGAGAGCTGTGGCTTAG 1458 1446 base pairs nucleic acid single linear cDNA 13ATGGCTAAGC TTGTTCTTTC CCTTTGTTTT CTGCTTTTCA GTGGCTGCTG CTTCGCTTTC 60AGTTTCAGAG AGCAGCCACA GCAAAACGAG TGCCAGATCC AACGCCTCAA TGCCCTAAAA 120CCGGATAACC GTATAGAGTC AGAAGGTGGC TTCATTGAGA CATGGAACCC TAACAACAAG 180CCATTCCAGT GTGCCGGTGT TGCCCTCTCT CGCTGCACCC TCAACCGCAA CGCCCTTCGC 240AGACCTTCCT ACACCAACGC TCCCCAGGAG ATCTACATCC AACAAGGTAG TGGTATTTTT 300GGCATGATAT TCCCGGGTTG TCCTAGCACA TTTGAAGAGC CTCAACAAAA AGGACAAAGC 360AGCAGGCCCC AAGACCGTCA CCAGAAGATC TATCACTTCA GAGAGGGTGA TTTGATTGCA 420GTGCCAACCG GTTTTGCATA CTGGATGTAC AACAATGAAG ACACTCCTGT TGTTGCCGTT 480TCTCTTATTG ACACCAACAG CTTCCAGAAC CAGCTCGACC AGATGCCTAG GAGATTCTAT 540CTTGCTGGGA ACCAAGAGCA AGAGTTTCTA CAGTATCAGC CACAGAAGCA GCAAGGAGGT 600ACTCAAAGCC AGAAAGGAAA GCGTCAGCAA GAAGAAGAAA ACGAAGGAGG CAGCATATTG 660AGTGGCTTCG CCCCGGAATT CTTGGAACAT GCGTTCGTCG TGGACAGGCA GATAGTGAGA 720AAGCTACAAG GTGAGAACGA AGAGGAAGAG AAGGGTGCCA TTGTGACAGT GAAAGGAGGT 780CTCAGCGTGA TAAGCCCACC CACGGAAGAG CAGCAACAAA GACCCGAGGA AGAGGAGAAG 840CCAGATTGTG ACGAGAAAGA CAAACATTGC CAAAGCCAAA GCAGAAATGG CATTGACGAG 900ACCATTTGCA CAATGAGACT TCGCCACAAC ATTGGCCAGA CTTCATCACC TGACATCTTC 960AACCCTCAAG CTGGTAGCAT CACAACCGCT ACCAGCCTCG ACTTCCCAGC CCTCTCGTGG 1020CTCAAACTCA GTGCCCAGTT TGGATCACTC CGCAAGAATG CTATGTTCGT GCCACACTAC 1080AACCTGAACG CAAACAGCAT AATATACGCA TTGAATGGAC GGGCATTGGT ACAAGTGGTG 1140AATTGCAATG GTGAGAGAGT GTTTGATGGA GAGCTGCAAG AGGGACAGGT GTTAATTGTG 1200CCACAAAACT TTGCGGTGGC TGCAAGATCA CAGAGCGACA ACTTCGAGTA TGTTTCATTC 1260AAGACCAATG ATAGACCCTC GATCGGCAAC CTTGCAGGTG CAAACTCATT GTTGAACGCA 1320TTGCCGGAGG AAGTGATTCA GCAAACTTTT AACCTAAGGA GGCAGCAGGC CAGGCAGGTC 1380AAGAACAACA ACCCTTTCAG CTTCCTGGTT CCACCTAAGG AGTCTCAGAG GAGAGTTGTG 1440GCTTAG 1446 1689 base pairs nucleic acid single linear cDNA 14ATGGGGAAGC CCTTCACTCT CTCTCTTTCT TCCCTTTGCT TGCTACTCTT GTCGAGTGCA 60TGCTTTGCTA TTAGCTCCAG CAAGCTCAAC GAGTGCCAAC TCAACAACCT CAACGCGTTG 120GAACCCGACC ACCGCGTTGA GTTCGAAGGT GGTTTGATTC AAACATGGAA CTCTCAACAC 180CCTGAGCTGA AATGCGCCGG TGTCACTGTT TCCAAACTCA CCCTCAACCG CAATGGCCTC 240CACTTGCCAT CTTACTCACC TTATCCCCGG ATGATCATCA TCGCCCAAGG GAAAGGAGCA 300CTGCAGTGCA AGCCAGGATG TCCTGAGACG TTTGAGGAGC CACAAGAACA ATCAAACAGA 360AGAGGCTCAA GGTCGCAGAA GCAGCAGCTA CAGGACAGTC ACCAGAAGAT TCGTCACTTC 420AATGAAGGAG ACGTACTCGT GATTCCTCCT GGTGTTCCTT ACTGGACCTA TAACACTGGC 480GATGAACCAG TTGTTGCCAT CAGTCTTCTT GACACCTCTA ACTTCAATAA CCAGCTTGAT 540CAAACCCCTA GGGTATTTTA CCTTGCTGGG AACCCAGATA TAGAGTACCC AGAGACCATG 600CAACAACAAC AACAGCAGAA AAGTCATGGT GGACGCAAGC AGGGGCAACA CCAGCAGGAG 660GAAGAGGAAG AAGGTGGCAG CGTGCTCAGT GGCTTCAGCA AACACTTCTT GGCACAATCC 720TTCAACACCA ACGAGGACAT AGCTGAGAAA CTTCAGTCTC CAGACGACGA AAGGAAGCAG 780ATCGTGACAG TGGAAGGAGG TCTCAGCGTT ATCAGCCCCA AGTGGCAAGA ACAACAAGAT 840GAAGATGAAG ATGAAGACGA AGATGATGAA GATGAACAAA TTCCCTCTCA CCCTCCTCGC 900CGACCAAGCC ATGGAAAGCG TGAACAAGAC GAGGACGAGG ACGAAGATGA AGATAAACCT 960CGTCCTAGTC GACCAAGCCA AGGAAAGCGT GAACAAGACC AGGACCAGGA CGAGGACGAA 1020GATGAAGATG AAGATCAACC TCGCAAGAGC CGCGAATGGA GATCGAAAAA GACACAACCC 1080AGAAGACCTA GACAAGAAGA ACCACGTGAA AGAGGATGCG AGACAAGAAA CGGGGTTGAG 1140GAAAATATCT GCACCTTGAA GCTTCACGAG AACATTGCTC GCCCTTCACG CGCTGACTTC 1200TACAACCCTA AAGCTGGTCG CATTAGTACC CTCAACAGCC TCACCCTCCC AGCCCTCCGC 1260CAATTCCAAC TCAGTGCCCA ATATGTTGTC CTCTACAAGA ATGGAATTTA CTCTCCACAT 1320TGGAATCTGA ATGCAAACAG TGTGATCTAT GTGACTCGAG GACAAGGAAA GGTTAGAGTT 1380GTGAACTGCC AAGGGAATGC AGTGTTCGAC GGTGAGCTTA GGAGGGGACA ATTGCTGGTG 1440GTACCACAGA ACTTCGTGGT GGCGGAGCAA GCCGGAGAAC AAGGATTCGA ATACATAGTA 1500TTCAAGACAC ACCACAACGC AGTCACTAGC TACTTGAAGG ATGTGTTTAG GGCAATTCCC 1560TCAGAGGTTC TTGCCCATTC TTACAACCTT CGACAGAGTC AAGTGTCTGA GCTTAAGTAT 1620GAAGGAAATT GGGGTCCTTT GGTCAACCCT GAGTCTCAAC AAGGCTCACC CCGTGTTAAA 1680GTCGCATAA 1689 1551 base pairs nucleic acid single linear cDNA 15ATGGGGAAGC CCTTCTTCAC TCTCTCTCTT TCTTCCCTTT GCTTGCTACT CTTGTCGAGT 60GCATGCTTTG CTATTACCTC CAGCAAGTTC AACGAGTGCC AACTCAACAA CCTCAACGCG 120TTGGAACCCG ACCACCGCGT TGAGTCCGAA GGTGGTCTTA TTGAAACATG GAACTCTCAA 180CACCCTGAGC TGCAATGCGC CGGTGTCACT GTTTCCAAAC GCACCCTCAA CCGCAACGGC 240TCCCACTTGC CATCTTACTT ACCTTATCCC CAAATGATCA TTGTCGTTCA AGGGAAGGGA 300GCAATTGGAT TTGCATTTCC GGGATGTCCC GAGACGTTTG AGAAGCCACA ACAACAATCA 360AGCAGAAGAG GCTCAAGGTC ACAGCAGCAA CTACAAGACA GTCACCAGAA GATTCGTCAC 420TTCAATGAAG GAGACGTACT AGTGATTCCT CTTGGTGTTC CTTACTGGAC CTATAACACT 480GGCGATGAAC CAGTTGTTGC CATCAGTCCT CTTGACACCT CCAACTTCAA CAATCAGCTT 540GATCAAAACC CCAGAGTATT TTACCTTGCT GGGAACCCAG ATATAGAGCA TCCCGAGACC 600ATGCAACAAC AGCAGCAGCA GAAGAGTCAT GGTGGACGCA AGCAGGGGCA ACACCGACAG 660CAGGAGGAAG AAGGTGGCAG TGTGCTCAGT GGCTTCAGCA AACATTTCTT AGCACAATCC 720TTCAACACCA ACGAGGACAC AGCTGAGAAA CTTCGGTCTC CAGATGACGA AAGGAAGCAG 780ATCGTGACAG TGGAGGGAGG CCTCAGCGTT ATCAGCCCCA AGTGGCAAGA ACAAGAAGAC 840GAAGACGAAG ACGAAGACGA AGAATATGGA CGGACGCCCT CTTATCCTCC ACGACGACCA 900AGCCATGGAA AGCATGAAGA TGACGAGGAC GAGGACGAAG AAGAAGATCA ACCTCGTCCT 960GATCACCCTC CACAGCGACC AAGCAGGCCC GAACAACAAG AACCACGTGG AAGAGGATGT 1020CAGACTAGAA ATGGGGTTGA GGAAAATATT TGCACCATGA AGCTTCACGA GAACATTGCT 1080CGCCCTTCAC GTGCTGACTT CTACAACCCA AAAGCTGGTC GCATTAGCAC CCTCAACAGT 1140CTCACCCTCC CAGCCCTCCG CCAATTCGGA CTCAGTGCCC AATATGTTGT CCTCTACAGG 1200AATGGAATTT ACTCTCCAGA TTGGAACTTG AACGCGAACA GTGTGACGAT GACTCGAGGG 1260AAAGGAAGAG TTAGAGTGGT GAACTGCCAA GGGAATGCAG TGTTCGACGG TGAGCTAAGG 1320AGGGGACAAT TGCTAGTGGT GCCGCAGAAC CCCGCGGTGG CTGAGCAAGG GGGAGAACAA 1380GGATTGGAAT ATGTAGTGTT CAAGACACAC CACAACGCCG TGAGCAGCTA CATTAAGGAT 1440GTGTTTAGGG TAATCCCTTC GGAGGTTCTT TCCAATTCTT ACAACCTTGG CCAGAGTCAA 1500GTGCGTCAGC TCAAGTATCA AGGAAACTCC GGCCCTTTGG TCAACCCATA A 1551 27 basepairs nucleic acid single linear DNA (genomic) 16 GCGGCCGCAT GGCCAAGCTAGTTTTTT 27 23 base pairs nucleic acid single linear DNA (genomic) 17GCGGCCGCTG GTGGCGTTTG TGA 23 23 base pairs nucleic acid single linearDNA (genomic) 18 GCGGCCGCTC TTCTGAGACT CCT 23 24 base pairs nucleic acidsingle linear DNA (genomic) 19 GCGGCCGCAT GCCCTTCACT CTCT 24 26 basepairs nucleic acid single linear DNA (genomic) 20 GCGGCCGCTG GGAGGGTGAGGCTGTT 26 24 base pairs nucleic acid single linear DNA (genomic) 21GCGGCCGCTG AGCCTTGTTG AGAC 24 12 base pairs nucleic acid single linearDNA (genomic) 22 ATAGCCCCCC AA 12

What is claimed is:
 1. A soybean plant transformed at a single locus inits genome with a chimeric gene comprising at least a portion of aglycinin or a beta conglycinin gene wherein said transformation resultsin reduction of the amount of at least one soybean seed storage protein,selected from the group consisting of glycinin and beta-conglycinin, inseed obtained from said transformed plant when compared to the amount ofsoybean seed storage protein in seed obtained from a non-transformedplant.
 2. Seeds obtained from the plant of claim 1.